Good prognosis of adult hemophagocytic lymphohistiocytosis associated with the germline HAVCR2 mutation.

Hemophagocytic lymphohistiocytosis (HLH) in adults may be idiopathic or secondary to various conditions. Recent studies identified germline hepatitis A virus-cellular receptor 2 (HAVCR2) mutations in subcutaneous panniculitis-like T-cell lymphoma (SPTCL) with HLH. The roles of this mutation in HLH, especially in idiopathic group, have never been explored. Of the 65 HLH cases, we detected germline HAVCR2Y82C mutations in nine (13.8%) cases (five SPTCL and four idiopathic HLH). Other causes of HLH were hematologic malignancies excluding SPTCL (32.3%), idiopathic HLH without HAVCR2 mutation (29.2%), infections (15.3%), and autoimmune diseases (9.2%). Germline HAVCR2 mutation was significantly associated with less anemia and better survival. This defines a distinct subgroup of HLH.

Stability of Hemoglobin Constant Spring Identified by Capillary Electrophoresis.

OBJECTIVE: Hemoglobin Constant Spring (HbCS) is often missed by routine hemoglobin analysis. The aim of this research was to study HbCS stability as identified by capillary electrophoresis (CE) to determine the specimen storage time limit. METHODS: The EDTA blood of 29 HbCS samples were kept at 4°C and analyzed every workday until CE could not detect HbCS or until 7 weeks after blood collection. The genotypes were confirmed by multiplex polymerase chain reaction. RESULTS: The median subject age was 27 years and 10 subjects were male. The HbCS levels were stable during the first 7 days but became undetectable in 5 cases (17.2%) after 1 week. All of them were heterozygous HbCS. Longer detection times were correlated with the higher baseline HbCS levels, with a correlation coefficient of 0.582 (P  ≤ 0.001). CONCLUSION: Routine hemoglobin typing and quantitation should be performed within 1 week after blood collection to detect low HbCS levels, especially in heterozygous HbCS.

Comprehensive screening for coexisting heterozygous α0-thalassemia in hemoglobin E trait.

Objectives: A sensitive screening for the coexistence of α0-thalassemia and the hemoglobin E (Hb E) trait is important to identify at-risk couples for hydrops fetalis. However, previous cutoff values have shown a positive predictive value (PPV) of only 50% or less. This study aimed to define more specific indicators to reduce the need for DNA tests. Methods: Patients with Hb E trait, as diagnosed by high performance liquid chromatography (HPLC) and/or isoelectric focusing (IEF) techniques, were tested for α0-thalassemia and α+-thalassemia deletions using multiplex gap polymerase chain reaction. Iron deficiency anemia (IDA) were excluded using a red cell distribution width (RDW) of more than 14.5%. Results: From 390 specimens, suitable cutoff values showing a 100% sensitivity for detection of heterozygous α0-thalassemia were an Hb E level of less than 22% by HPLC, a mean corpuscular volume (MCV) of less than 72 fL, and a mean corpuscular hemoglobin (MCH) level of less than 22.5 pg. Comparable results were obtained in the validation cohort (N = 179). Using a combination of Hb E with either MCV or MCH cutoff points gave a PPV of 76.2% and 77.4%, respectively. Discussion: IDA was reported to interfere with Hb E level. In this study, we excluded IDA using RDW of more than 14.5% to enhance the test specificity. Conclusion: Lower cutoff screening values can be used to exclude α0-thalassemia in the Hb E trait yielding a higher specificity in a normal RDW condition. This can save the cost and labor of DNA testing.

Natural anticoagulant deficiencies in Thais: A population-based study.

INTRODUCTION: In contrast to Caucasians, hereditary anticoagulant deficiencies are more common in Asians and mutations are heterogeneous among different countries. This study aimed to determine the prevalence and genetic basis of protein C and protein S deficiencies in Thai population. METHODS: Healthy volunteers were tested for protein C activity and free protein S antigen. Subjects with low values were requested for repeated testing and exclusion of acquired causes. Cases with persistently low levels were assayed for respective gene mutations using direct sequencing and multiplex ligation-dependent probe amplification (MPLA) when PROS1 point mutation was undetectable. RESULTS: For protein C activities (N = 5234), the values of men were lower than those of post-menopausal women (P < 0.001). In 17 of 18 subjects, there were 7 types of PROC mutations, 64.7% of which were p.R189W and 2 were previously unreported. Protein S levels (N = 5242) were highest in men, followed by post-menopausal and pre-menopausal women, respectively (P < 0.001). The repeatedly low protein S was mostly in female (88.2%). Among 29 subjects with protein S below the sex-specific 2.5 percentile cut-points, 4 types of mutations were found in 5 subjects (17.2%) with one previously unreported mutation. Free protein S levels were below 30 U/ml in all cases with mutations. The estimated prevalence of PROC and PROS1 mutations in Thai population was 0.69% and 0.22%, respectively. CONCLUSIONS: PROC mutations, especially p.R189W, are common in Thais. However, mildly depressed protein S levels were frequently found in women with undetectable PROS1 mutation.

The first validated criteria for effective screening and a new simplified method for α-globin gene sequencing for diagnosis of uncommon α-globin mutations.

No well-defined phenotypes that distinguish between unknown α- and ß-globin mutations have been reported to date. Direct DNA sequencing of α-globin genes can be technically challenging, as α1- and α2-globin genes are nearly indistinguishable. To detect hemoglobin variants (HbXs) on Hb analysis, the entire ß- and α-globin genes were directly sequenced using a newly developed sequencing protocol for α-globin genes. An algorithm to distinguish between α- and ß-HbXs was constructed and subsequently validated in the independent validation group. Distinctive characteristics that can distinguish 39 α-HbXs from 24 ß-HbXs were the presence of unidentifiable variants of HbA2 and/or HbX of <37% on isoelectric focusing and <31% on high-performance liquid chromatography. Another set of 67 HbXs was employed to validate our algorithm. This accurately predicted 33 α-HbXs with 100% sensitivity and 97.1% specificity. Our sequencing protocol for α-globin genes was able to identify 11 rare mutations among all exons of both α-globin genes from 72 subjects. Six of these variants were first discovered in Thais. This is the first well-characterized algorithm for distinguishing unknown Hb variants in a large cohort. Our validated criteria and DNA sequencing procedure are highly efficient for molecular characterization of rare Hb mutations.

Diagnostic utility of isoelectric focusing and high performance liquid chromatography in neonatal cord blood screening for thalassemia and non-sickling hemoglobinopathies.

BACKGROUND: Thalassemia syndromes are highly prevalent in Southeast Asia. In Thailand, high performance liquid chromatography (HPLC) is the most common technique routinely performed in diagnosis of thalassemia and hemoglobinopathies, while isoelectric focusing (IEF) is rarely employed. We compared the diagnostic utility of IEF and HPLC in neonatal screening for thalassemia and non-sickling hemoglobinopathies. METHODS: Two-hundred and forty-one cord blood samples were analyzed using IEF and HPLC, ß-thalassemia short program. The results were correlated with red cell indices and molecular analyses. Hemoglobin (Hb) Bart's was quantified only on IEF. RESULTS: Of 241 newborns, IEF and HPLC yielded 85.4% and 76.4% sensitivity to identify α-thalassemia syndrome, respectively. HbBart's≥2% yielded 100% sensitivity to identify 2 α-globin gene deletions and/or mutations, while MCV≤95fl and MCH≤30pg yielded 100% sensitivity to identify 2 α-globin gene deletions. DNA analysis revealed HbE mutation in all 61 subjects with HbA2>1% on both IEF and HPLC. CONCLUSION: IEF is an effective method in neonatal screening for thalassemia and non-sickling hemoglobinopathies. The HbBart's level, MCV and MCH are helpful for identifying α-thalassemia. The presence of HbA2 higher than 1% in cord blood indicates HbE carriers in Southeast Asian newborns.

Clinical and hematological characteristics of uncommon beta-globin variants in Thailand.

Hemoglobin (Hb) E is the commonest beta-globin variant in Thailand. Other uncommon variants have been rarely reported. We performed direct DNA sequencing of the entire beta-globin gene in cases showing unidentified bands by isolectric focusing electrophoresis or high-performance liquid chromatography. Six different beta-globin mutations were found in 24: 9 Hb Hope, 6 Hb New York, 4 Hb Tak, 3 Hb D Punjab, 1 Hb Pyrgos, and 1 Hb Rambam. Hb New York was misidentified as HbA on HPLC. All Hb Tak carriers had hemoglobin over 15.0 g/dL and a Hb Tak/beta(0)-thalassemia compound heterozygote presented with symptomatic polycythemia.

Elevated serum transferrin receptor levels in common types of thalassemia heterozygotes in Southeast Asia: a correlation with genotypes and red cell indices.

BACKGROUND: Serum transferrin receptor (sTfR) measurement is a helpful test for diagnosis of iron deficiency. Increased values are detectable in thalassemia syndromes due to increased erythropoiesis. However, sTfR has never been studied in hemoglobin E (HbE) carriers and their interactions with alpha-thalassemia heterozygotes that are common in Southeast Asia. METHODS: We determined sTfR concentrations using a particle enhanced immunoturbidimetric assay in 113 early pregnancies without iron deficiency. RESULTS: Patients were genotypically classified into 6 groups: 23 normal (mean sTfR+/-SD mg/l, 0.94+/-0.22), 14 alpha(+)-thalassemia heterozygotes (1.06+/-0.45), 21 alpha(0)-thalassemia heterozygotes (1.31+/-0.35), 30 HbE heterozygotes (1.11+/-0.26), 13 HbE heterozygotes with alpha(+)-thalassemia heterozygotes (1.09+/-0.32), and 12 HbE heterozygotes with alpha(0)-thalassemia heterozygotes (1.16+/-0.27). sTfR concentrations in all thalassemic groups were higher than controls, and significantly correlated with high red cell count, low MCV and MCH (p<0.001). When alpha(0)- or alpha(+)-thalassemia combined with HbE, sTfR concentrations were declined compared with alpha(0)-thalassemia or hemoglobin E, respectively, suggesting more balances in alpha- and beta-globin chain production. CONCLUSIONS: Mildly increased erythropoiesis represented by increased sTfR concentrations in alpha-thalassemia and HbE heterozygotes and illustrated alpha- and beta-thalassemic gene interaction. These findings warrant further investigations on sTfR in diagnosis of iron deficiency in thalassemia carriers.

Severe beta(0) thalassemia/hemoglobin E disease caused by de novo 22-base pair duplication in the paternal allele of beta globin gene.

beta Thalassemia is a major public health concern in Southeast Asia. A prevention program has been implemented in Thailand comprising mass carrier screening and genetic testing. In this study, a Thai girl with severe beta thalassemia/hemoglobin (Hb) E disease was born from the mother with Hb E trait and the genotypically normal father. DNA sequencing revealed novel 22-bp tandem duplication in the paternal allele of beta globin gene, producing a severely truncated product. A short recurring nucleotide at the insertion site suggested a predisposition to this mutation. Therefore, spontaneous beta globin mutations occasionally occur in normal population. Its clinical significance is noteworthy in countries with high prevalence of beta thalassemia.

Evaluation of automated glycohemoglobin analyzer HLC-723G7 beta-thalassemia analysis mode.

We evaluated the new automated glycohemoglobin analyzer HLC-723G7 (G7) intended for beta-thalassemia diagnosis to determine correlation of hemoglobin (Hb) F and Hb A2 quantitation with the Variant beta-Thalassemia Short Program and to evaluate the performance of the G7 analyzer. Two hundred fifty EDTA blood samples with Hb A, A2, and F by Variant were analyzed for Hb A2 and Hb F by G7 device. Recovery, precision, and interference of the G7 analyzer were studied. The R2 values for Hb A2 and Hb F were 0.963 and 0.640, respectively. Recovery of Hb A2 and Hb F of low and high control samples ranged from 96% to 100% and 91% to 101%, respectively. The coefficients of variation (CVs) of intraassay precision ranged from 1.3% to 3.9% for Hb F and 0.0 to 1.9% for Hb A2. The CVs of interassay precision ranged from 1.9% to 5.6% for Hb F and 2.5% to 3.0% for Hb A2. The CVs for Hb F in both conjugated bilirubin and chyle substance groups were higher than the CVs for Hb A2. This evaluation revealed very good correlation of Hb A2 measurement between the G7 and the Variant devices. The performance evaluation demonstrated good recovery, precision, and low interference of both Hb A2 and Hb F measurements.